Isolation and structure of cribrostatin 6

ABSTRACT

Cribrostatin 6, a dark blue cancer cell growth inhibiting constituent of the Republic of Maldives marine sponge Cribrochalina sp. has been isolated, and its structure (shown below) elucidated, based on a combination of RMS, high field (500 MHz, HMBC, and GOESY experiments) 15N,  1 H- and 13C NMR, and X-ray crystal structure analyses. Cribrostatin 6 also was found to inhibit the growth of a number of pathogenic bacteria and fungi.

RELATED APPLICATION DATA

This application is based on and claims the benefit of U.S. ProvisionalPatent Application No. 60/449,167 filed on Feb. 20, 2003.

INTRODUCTION

Financial assistance for this invention was provided by the UnitedStates Government, Division of Cancer Treatment and Diagnosis, NationalCancer Institute, Department of Health and Human Services OutstandingInvestigator Grant Numbers R01-CA9044-01 and CA44344-05-1-12; theArizona Disease Control Research Cornmission; and private contributions.Thus, the United States Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the isolation from natural sources, andelucidation of the structure of, a compound having antineoplastic,antibacterial and antifungal properties.

BACKGROUND OF THE INVENTION

Marine porifera have continued to be an increasingly important source ofnew nitrogen heterocyclic compounds with significant biologicalactivities. Recent examples include the cytotoxic constituents pateomine(Mycale sp.), a pyridine betaine (Microcosnus vulgaris), topsentin B2(Rhaphisia lacazei), asmarine A (Raspailia sp.), cyclic guanidines(Monanchiora sp.), the antiviral dragmacidin F (Halicortex sp.) and theisolation and structure determination of cribrostatins 4 (1) and 5 (2)from the Republic of Maldives blue-colored sponge Cribroclhalina sp.(West, L.; et al., J. Org. Chem. 2000, 65, 445-449; Aiello, A., et al.,J. Nat. Prod. 2000, 63, 517-519; Casapullo, A., et al., J. Nat. Prod.2000, 63, 447-451; Yosief, T., et al., J. Nat. Prod. 2000, 63, 299-304;Braekman, J., et al., J. Nat. Prod. 2000, 63, 193-196; Cutignano, A., etal., Tetrahedron 2000, 56, 3743-3748, Pettit, G., et al., J. Nat. Prod.2000, 6, 793-798.)

SUMMARY OF THE INVENTION

The present invention relates to the elucidation of the molecularstructure for a novel compound denominated Cribrostatin 6, as well as toa method for isolating the compound Cribrostatin 6 from the Marineorganism Cribrochalina sp. Cribrostratin 6 exhibits antineoplastic,antibacterial and antiflngal properties. Accordingly, the invention alsorelates to the use of Cribrostatin 6 as a pharmaceutical agent for thetreatment of neoplastic disease, as well as for the treatment ofbacterial and fungal infections.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the solid-state structure of Cribrostatin 6.

FIG. 2 illustrates the chemical structures of Cribrostatins 1, 2, 3, 4,5 and 6.

DETAILED DESCRIPTION OF THE INVENTION

Earlier we had observed a number of biologically active blue to blackcolored fractions arising during P388 lymphocytic leukemia guidedseparations of a 195 g dichloromethane-soluble portion of the extractobtained from 350 kg (wet wt.) of Cribrochalina sp. The cancer cellgrowth (P-388) inhibitory dark-colored fractions were finally separatedby a successive series of gel permeation and partition chromatographictechniques on Sephadex LH-20. That sequence was followed by high-speedcountetcurrent distribution using an Ito Coil-Planet centrifuge toafford 88 mg of a dark-blue constituent (P388 ED₅₀ 0.3 μg/ml),designated cribrostatin 6 (3). Owing to difficulties in unequivocallydeducing the structure of this interesting substance based on spectralevidence, attempts were made at various times over a ten year period toreach a correct solution and/or to produce crystals suitable for X-raystructure determination. We were eventually pleased to find thatcribrostatin 6 (3) would crystallize from acetone following long coldstorage of the solution. To follow is a summary of the spectral andX-ray crystallographic interpretation that completed a correctstructural assignment for cribrostatin 6 (3).

Results and Discussion

The molecular formula of cribrostatin 6 (3) was established asC₁₅H₁₄N₂O₃ by HRMS, using an APCI inlet system. Inspection of the ¹H andAPT NMR spectra indicated the presence of three methyls, one methylene,three methines and eight quaternary carbons. Protonated carbons wereassigned using a HMQC experiment. The APT spectrum indicated that fourof the carbons were oxygenated and suggested the presence of thequinone. An HMBC experiment allowed placement of the C-9 ethoxy (H-13 toC-9) and C-8 methyl (H-12 to C-7, 8, and 9) groups and established thepositions of the quaternary carbons at C-8 and 9 as well as the carbonylcarbons at C-7 and 10, which were assigned by analogy with knownisoquinolinequinones such as the Saframycins. (Cooper, R., et al.,Antibiotics 1985, 38, 24-30). This accounted for five of the ten degreesof unsaturation determined from the molecular formula. The nature of theB ring was established by 1H-1H COSY, which indicated the presence of adouble bond. The HMBC spectrum showed connectivities from the proton atH-6 to C-6a, 7 and 10a as well as establishing the position of thedouble bond at Δ, which was confirmed by HMBC correlations from H-5 toC-6 and 6a. (Braekman, J., et al., J. Nat. Prod. 2000, 63, 193-196;Cutignano, A., et al., Tetrahedron 2000, 56, 3743-3748.) The remainingthree degrees of unsaturation and the fragment C₃H₄N₂ suggested animidazo-partial structure for a C ring. The overall structure wasdetermined by X-Ray diffraction on a small needle-shaped crystal.Although the overall connectivity could be readily established, the lowobserved data-to-parameter ratio did not permit a clear distinctionbetween structures 3 and 4. Analogy to previous cribrostatin-relatedcompounds (cf. 1, 2) gave the location of one of the N atoms at position4 with reasonable certainty (Pettit, G., et al., J. Nat. Prod. 2000, 6,793-798; Pettit, G., et al., Can. J. Chem. 1992, 70, 1170-1175), butconclusive structural assignment as9-ethoxy-3,8-dimethyl-imidazo[5,1-a]isoquinoline-7, 10-dione (3)required further, more detailed analysis of earlier and new NMR data.

Examination of the HMBC spectrum showed correlations from H5 to C-3 andC10b and implied placement of a nitrogen at position 4. A strongcorrelation from the remaining methine proton to C-3 suggested position1 with the remaining nitrogen at position 2. An additional correlationfrom H-11 to C-3 located the remaining methyl group (δ 2.75p) at C-3. ADPFGSENOE (GOESY) experiment demonstrated NOE enhancement between H-5and H-11 that would be consistent with either structure 3 or 4, but gaveno indication of an enhancement between H-2 and H-11, that would beexpected to exist in structure 4 (Stonehouse, J., et al., J. Amer. Chem.Soc. 1994, 116, 6037-6038). Measurement of ¹⁵N-¹H HMBC showed two strong3-bond correlations from the methyl protons H11 to both nitrogens N2 andN4. HMBC correlations were observed H5 and H6 to N4, but not N2. H1showed weak correlations to both N2 and N4. Only structure 3 isconsistent with these results.

In addition to cancer cell growth inhibition of murine P388 lymphocyticleukemia and human cancer cell lines (see Table II), cribrostatin 6exhibited antimicrobial activity against numerous antibiotic-resistantGram-positive bacteria and patlhogenic fungi (see Table III). The onlyGram-negative bacterium of those tested which was inhibited bycribrostatin 6 was Neisseria gonorrhoeae. Cribrostatin 2 has anantimicrobial profile similar to cribrostatin 6, while cribrostatins 1,3, 4 and 5 have antibacterial but not antifungal activities. (Pettit,G., et al., J. Nat. Prod. 2000, 6, 793-798). Thus, the inventors believethat the cribrostatins, particularly cribrostatin 6, warrant furtherinvestigation as antibacterial and/or antifungal agents.

Recently, two phosphorylated sterol sulfates were isolated from aCribrochalina sp. and found to be membrane-type metalloproteinase(MT1-MMP) inhibitors. (Fujita, M., et al., Tetrahedron 2001, 57,3885-3890.) That advance extends the structural variety of Cribrochalinagenus cell growth regulatory constituents that so far range fromacetylenic alcohols to quinones (cf. 3) and peptides. (Hallock, Y., etal., J. Nat. Prod. 1995, 58, 1801-1807; Garcia, J., et al. Tetrahedron:Asymmetry 1999, 10, 2617-2626; Sharma, A.; et al., S. Tetrahedron:Asymmetry 1998, 9, 2635-2639; Pettit, G., et al., J. Nat. Prod. 2000, 6,793-798; Pettit, G., et al., Can. J Chem. 1992, 70, 1170-1175; Yeung,B., et al., J. Org. Chem. 1996, 61, 7168-7173.)

Experimental Section

General Experimental Methods. Except as noted, the general experimentalprocedures employed in our original investigations of the Cribrochalinasp. were continued here. For discussion of these originalinvestigations, see Pettit, G., et al., J. Nat. Prod. 2000, 6, 793-798;and Pettit, G., et al., Can. J Chem. 1992, 70, 1170-1175, which areincorporated herein by reference. NMR spectra were recorded using aVarian Inova system equipped with a 5 mm triple resonance triaxial PFGprobe at 500 MHz for ¹H and 125 MHz for ¹³C, and 50.65 MHz for ¹⁵N.¹⁵N-¹H gradient HMBC experiments were performed on 2.2 mg of sampledissolved in 100 μl CDCl₃ using a Shigemi 3 mm NMR tube susceptibilitymatched to CDCl₃, a Nalorac 3 mm ¹H{¹⁵N-³¹P} indirect-detection probeand delays optimized for coupling constants of 90 Hz (1-bond) and 5 Hz(multiple-bond). The ¹⁵N spectra were referenced to formamide (112 pmdownfield of liquid ammonia). (Martin, G. et al., J. Nat. Prod. 2000,63, 543-585.) The ¹H NMR and ¹³C NMR spectra were referenced to residualsolvent signals at 7.25 and 77.0 ppm for CDCl₃. HRMS data was obtainedusing a JEOL LCMate magnetic sector instrument in the APCI mode,calibrated using a polythylene glycol reference mixture. The X-Ray datacollection was accomplished using a Bruker AXS 6000 diffractometer.

Isolation of Cribrostatin 6 (1). The blue marine sponge Cribrochalinasp. was collected and extracted as known to one of skill in the art, asdescribed in Pettit, G., et al., J. Nat. Prod. 2000, 6, 793-798 andPettit, G., et al., Can. J. Chem. 1992, 70, 1170-1175, which isincorporated herein by reference. Fractionation of the extract, guidedby the blue color, and the screening results obtained using the murineP-388 lymphocytic leukemia cell line, was carried out on columns ofSephadex LH-20, eluted successively with a.) CH₃OH; b.) CH₂Cl₂-CH₃OH(3:2); c.) hexane-toluene-CH₃OH (3:1:1); and d.) hexane/i-PrOH-CH₃OH(8:1:1). In preparation for a separation using high-speed countercurrentdistribution on an Ito Coil-Planet centrifuge, the blue fraction fromthe previous column was triturated with the upper (less polar) phase ofthe system hexane-EtOAc-CH₃OH-water (700:300:150:60), and the solutionwas filtered. The sparingly-soluble material thus obtained (35.7 mg)proved to be the same as the solid isolated from the principal bluefraction from the countercurrent run (53 mg). The two were combined andrecrystallized from acetone to afford dark-blue needles: mp 169-171° C.;P-388 ED₅₀ 0.3 μg/ML; λ_(max) 203 (26,758), 266 (24,432), 323 (5597),552 (1479); IR v_(max) 2920, 1660, 1620, 1605, 1522, 1170 cm⁻¹; ¹H and¹³C-NMR, see Table I; LREIMS (m/z) 270, 242, 214, 185, 172, 157, 145,116; HRMS (APCI⁺) 271.10968 (calcd for (M+H)⁺ ion C₁₅H₁₅N₂O₃, 271.10828error 5.2 ppm).

Crystal Structure of Cribrostatin 6 (3). All data including atomiccoordinates, thermal parameters, bond distances, angles, and observedand calculated structure factors have been deposited in the CambridgeCrystallographic Data Centre and can be obtained, free of charge, onapplication to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK(fax: +44-(0)1223-336003 or e-mail: deposit@ccdc.cam.ac.uk). A verysmall, dark-blue needle obtained via slow evaporation of an acetonesolution, with approximate dimensions of (0.05×0.05×0.20 mm), wasmounted on the tip of a glass fiber. An initial set of cell constantswas calculated from reflections harvested from three sets of 60 framesat 298(2) K on a Bruker 6000 diffractometer. Cell parameters indicatedan orthorhombic space group. Subsequent data collection, using 30 secondscans/frame and 0.396° steps in Ω, was conducted in such a manner as tocompletely survey a complete hemisphere of reflections. This resultedin >93% coverage of the total reflections possible to a resolution of0.83. A total of 10229 reflections were harvested from the total datacollection and final cell constants were calculated from a set of 332strong, unique reflections. Subsequent statistical analysis of thecomplete reflection data set using the XPREP program indicated the spacegroup was Pca2₁. The XPREP program is an automatic space determinationprogram included in the SHELXTL-NT-Version 5.10 (1997), which anintegrated suite of programs for the determination of crystal structuresfrom diffraction data, that is available from Bruker AXS, Inc., Madison,Wis. 53719, USA. This package includes, among others, XPREP, SHELXS (astructure solution program via Patterson or direct methods), and SHELXL(structure refinement software).

Crystal data: C₁₅H₁₄N₂O₃, a=15.414(15), b=11.532(11), c=7.201(7) Å,V=1280(2) Å³, λ=(Cu Kα)=1.54178 Å, μ (Cu K)=0.817 mm⁻¹, ρc=1.403 g cm⁻³for Z=4 and M_(r)=270.28, F (000)=568. After data reduction, merging ofequivalent reflections and rejection of systematic absences, 1885 uniquereflections remained (R_(int)=0.5248), of which 315 were consideredobserved (I_(o)>2 (I_(o))) and were used in the subsequent structuresolution and refinement. An absorption correction was applied to thedata with SADBS. (Blessing, R., Acta Cryst., 1995, A51, 33-8.) Directmethods structure determination and refinement were accomplished withthe SHELXTL NT ver.V5.10 suite of programs. All non-hydrogen atoms forcribrostatin 6 (3) were located using the default settings of thatprogram. Although the overall connectivity of the non-hydrogen atoms inquinone 3 could be readily established from the X-ray data, the lowobserved data-to-parameter ratio did not allow a completely unambiguousassignment of the two nitrogen atoms. The location of one of the N atomsat position 9 (FIG. 1; X-ray numbering system) was known with reasonablecertainty (due to analogy to previous cribrostatin related compounds),the position of the second N atom was less certain, with positions 11and 12 both being likely candidates. Refinement of each of thesepossible isomeric structures (i.e., structures 3 or 4) resulted innearly identical residual R₁ values (0.0982 vs 0.1002, respectively).Although the former (3) was slightly favored by these results, thefinal, conclusive structural assignment was based on observed ¹⁵N-NMRexperiments. Since the quality of data precluded the directdetermination of hydrogen atom positions, the remaining hydrogen atomcoordinates were calculated at optimum positions using the programSHELXL. These latter atoms were assigned thermal parameters equal toeither 1.2 or 1.5 (depending upon chemical type) of the Uiso value ofthe atom to which they were attached, then both coordinates and thermalvalues were forced to ride that atom during final cycles of refinement.All non-hydrogen atoms were refined anisotropically in a full-matrixleast-squares refinement process. The final standard residual R₁ valuefor the model shown in FIG. 1 was 0.0982 (for observed data) and 0.3817(for all data). The corresponding Sheldrick R values were wR₂ of 0.2174and 0.2741, respectively. The difference Fourier map showedinsignificant residual electron density; the largest difference peak andhole being +0.255 and −0.252 e/Å³, respectively. Final bond distancesand angles were all within acceptable limits.

Cancer Cell Growth Inhibition

Compounds were screened against a panel of human cancer cell lines andmouse cell lines as is shown in Table II. Cribrostatin 6 exhibitedcancer cell growth against all lines illustrated.

Antimicrobial Susceptibility Testing

Compounds were screened against bacteria and fungi according toestablished broth microdilution susceptibility assays, pursuant to theNational Committee for Clinical Laboratory Standards, Methods forDilution Antimicrobial Susceptibility Tests for Bacteria that GrowAerobically, Approved Standard M7-A4, Wayne, Pa.: NCCLS, 1997, and theNational Conmnittee for Clinical Laboratory Standards, Reference Methodfor Broth Dilution Antifungal Susceptibility Testing of Yeasts, ApprovedStandard M27-A, Wayne, Pa.: NCCLS, 1997. The results of such screeningare shown in Table III. The minimum inhibitory concentration was definedas the lowest concentration of compound that inhibited all visiblegrowth of the test organism (optically clear). Assays were repeated onseparate days.

ADMINISTRATION

Dosages

The dosage of the presently disclosed compounds to be administered tohumans and other animals requiring treatment will depend upon numerousfactors, including the identity of the neoplastic disease or microbialinfection; the type of host involved, including its age, health andweight; the kind of concurrent treatment, if any; the frequency oftreatment and therapeutic ratio. Hereinafter are described variouspossible dosages and methods of administration, with the understandingthat the following are intended to be illustrative only, and that theactual dosages to be administered, and methods of administration ordelivery may vary therefrom. The proper dosages and administration formsand methods may be determined by one of skill in the art.

Illustratively, dosage levels of the administered active ingredientsare: intravenous, 0.1 to about 20 mg/kg; intramuscular, 1 to about 50mg/kg; orally, 5 to about 100 mg/kg; intranasal instillation, 5 to about100 mg/kg; and aerosol, 5 to about 100 mg/k of host body weight.

Expressed in terms of concentration, an active ingredient can be presentin the compositions of the present invention for localized use about thecutis, intranasally, pharyngolaryngeally, bronchially, intravaginally,rectally, or ocularly in concentration of from about 0.01 to about 50%w/w of the composition; preferably about 1 to about 20% w/w of thecomposition; and for parenteral use in a concentration of from about0.05 to about 50% w/v of the composition and preferably from about 5 toabout 20% w/v.

The compositions of the present invention are preferably presented foradministration to humans and animals in unit dosage forms, such astablets, capsules, pills, powders, granules, suppositories, sterileparenteral solutions or suspensions, sterile non-parenteral solutions ofsuspensions, and oral solutions or suspensions and the like, containingsuitable quantities of an active ingredient. Other dosage forms known inthe art may be used.

For oral administration either solid or fluid unit dosage forms can beprepared.

Powders are prepared quite simply by comminuting the active ingredientto a suitably fine size and mixing with a similarly comminuted diluent.The diluent can be an edible carbohydrate material such as lactose orstarch. Advantageously, a sweetening agent or sugar is present as wellas a flavoring oil.

Capsules are produced by preparing a powder mixture as hereinbeforedescribed and filling into formed gelatin sheaths. Advantageously, as anadjuvant to the filling operation, a lubricant such as talc, magnesiumstearate, calcium stearate and the like is added to the powder mixturebefore the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurryof active ingredients with an acceptable vegetable oil, light liquidpetrolatum or other inert oil or triglyceride.

Tablets are made by preparing a powder mixture, granulating or slugging,adding a lubricant and pressing into tablets. The powder mixture isprepared by mixing an active ingredient, suitably comminuted, with adiluent or base such as starch, lactose, kaolin, dicalcium phosphate andthe like. The powder mixture can be granulated by wetting with a bindersuch as corn syrup, gelatin solution, methylcellulose solution or acaciamucilage and forcing through a screen. As an alternative to granulating,the powder mixture can be slugged, i.e., run through the tablet machineand the resulting imperfectly formed tablets broken into pieces (slugs).The slugs can be lubricated to prevent sticking to the tablet-formingdies by means of the addition of stearic acid, a stearic salt, talc ormineral oil. The lubricated mixture is then compressed into tablets.

Advantageously, the tablet can be provided with a protective coatingconsisting of a sealing coat or enteric coat of shellac, a coating ofsugar and methylcellulose and polish coating of carnauba wax.

Fluid unit dosage forms for oral administration such as in syrups,elixirs and suspensions can be prepared wherein each teaspoonful ofcomposition contains a predetermined amount of an active ingredient foradministration.

The water-soluble forms can be dissolved in an aqueous vehicle togetherwith sugar, flavoring agents and preservatives to form a syrup. Anelixir is prepared by using a hydroalcoholic vehicle with suitablesweeteners together with a flavoring agent. Suspensions can be preparedof the insoluble forms with a suitable vehicle with the aid of asuspending agent such as acacia, tragacanth, methylcellulose and thelike.

For parenteral administration, fluid unit dosage forms are preparedutilizing an active ingredient and a sterile vehicle, water beingpreferred. The active ingredient, depending on the form andconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the water-soluble active ingredient can bedissolved in water for injection and filter sterilized before fillinginto a suitable vial or ampule and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle. Parenteral suspensions are prepared insubstantially the same manner except that an active ingredient issuspended in the vehicle instead of being dissolved and sterilizationcannot be accomplished by filtration. The active ingredient can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive ingredient.

In addition to oral and parenteral administration, the rectal andvaginal routes can be utilized. An active ingredient can be administeredby means of a suppository. A vehicle which has a melting point at aboutbody temperature or one that is readily soluble can be utilized. Forexample, cocoa butter and various polyethylene glycols (Carbowaxes) canserve as the vehicle.

For intranasal installation, a fluid unit dosage form is preparedutilizing an active ingredient and a suitable pharmaceutical vehicle,preferably P.F. water, a dry powder, can be formulated when insulationis the administration of choice.

For use as aerosols, the active ingredients can be packaged in apressurized aerosal container together with a gaseous or liquefiedpropellant, for example, dichlorodifluoromethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable. Theterm “unit dosage form” as used in the specification and claims refersto physically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitation inherent in the art ofcompounding such an active material for therapeutic use in humans, asdisclosed in this specification, these being features of the presentinvention. Examples of suitable unit dosage forms in accord with thisinvention are tablets, capsules, troches, suppositories, powder packets,wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules,vials, segregated multiples of any of the foregoing, and other forms asherein described.

The active ingredients to be employed as antineoplastic and/orantimicrobial agents can be easily prepared in such unit dosage formwith the employment of pharmaceutical materials which themselves areavailable in the art and can be prepared by established procedures. Thefollowing preparations are illustrative of the preparation of the unitdosage forms of the present invention, and not as a limitation thereof.The following are examples of several dosage forms, in which thenotation “active ingredient” signifies Cribrostatin 6.

COMPOSITION “A” Hard-Gelatin Capsules

One thousand two-piece hard gelatin capsules for oral use, each capsulecontaining 200 mg of an active ingredient are prepared from thefollowing types and amounts of ingredients: Active ingredient,micronized 20 g Corn Starch 20 g Talc 20 g Magnesium stearate  2 g

The active ingredient, finely divided by means of an air micronizer, isadded to the other finely powdered ingredients, mixed thoroughly andthen encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

Using the procedure above, capsules are similarly prepared containing anactive ingredient in 5, 25, and 50 mg amounts by substituting 5 g, 25 gand 50 g of an active ingredient for the 20 g used above.

COMPOSITION “B” Soft Gelatin Capsules

One-piece soft gelatin capsules for oral use, each containing 20 mg ofan active ingredient, finely divided by means of an air micronizer, areprepared by first suspending the compound in 0.5 ml of corn oil torender the material capsulatable and then encapsulating in the abovemanner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

COMPOSITION “C” Tablets

One thousand tablets, each containing 20 mg of an active ingredient, areprepared from the following types and amounts of ingredients: Activeingredient, micronized 20 g Lactose 300 g Corn starch 50 g Magnesiumstearate 4 g Light liquid petrolatum 5 g

The active ingredient, finely divided by means of an air micronizer, isadded to the other ingredients and then thoroughly mixed and slugged.The slugs are broken down by forcing them through a Number Sixteenscreen. The resulting granules are then compressed into tablets, eachtablet containing 20 mg of the active ingredient.

The foregoing tablets are useful for treating a neoplastic disease bythe oral administration of one or two tablets one to four times a day.

Using the procedure above, tablets are similarly prepared containing anactive ingredient in 25 mg and 10 mg amounts by substituting 25 g and 10g of an active ingredient for the 20 g used above.

COMPOSITION “D” Oral Suspension

One liter of an aqueous suspension for oral use, containing in eachteaspoonful (5 ml) dose, 50 mg of an active ingredient, is prepared fromthe following types and amounts of ingredients: Active ingredient,micronized 1 g Citric acid 2 g Benzoic acid 1 g Sucrose 790 g Tragacanth5 g Lemon Oil 2 g Deionized water, q.s. 1000 ml

The citric acid, benzoic acid, sucrose, tragacanth and lemon oil aredispersed in sufficient water to make 850 ml of suspension. The activeingredient, finely divided by means of an air micronizer, is stirredinto the syrup unit uniformly distributed. Sufficient water is added tomake 1000 ml.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 teaspoonful (15 ml) three times a day.

COMPOSITION “E” Parenteral Product

A sterile aqueous suspension for parenteral injection, containing 3 mgof an active ingredient in each milliliter for treating a neoplasticdisease, is prepared from the following types and amounts ofingredients: Active ingredient, micronized 3 g POLYSORBATE 80 5 gMethylparaben 2.5 g Propylparaben 0.17 g Water for injection, q. s. 1000ml.

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair rnicronizer, and the final suspension is filled into sterile vialsand the vials sealed.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 milliliter (1 ml) three times a day.

COMPOSITION “F” Suppository, Rectal and Vaginal

One thousand suppositories, each weighing 2.5 g and containing 20 mg ofan active ingredient are prepared from the following types and amountsof ingredients: Active ingredient, micronized 1.5 g Propylene glycol 150g Polyethylene glycol #4000, q.s. 2,500 g

The active ingredient is finely divided by means of an air micronizerand added to the propylene glycol and the mixture passed through acolloid mill until uniformly dispersed. The polyethylene glycol ismelted and the propylene glycol dispersion is added slowly withstirring. The suspension is poured into unchilled molds at 40° C. Thecomposition is allowed to cool and solidify and then removed from themold and each suppository foil wrapped.

The foregoing suppositories are inserted rectally or vaginally fortreating a neoplastic disease.

COMPOSITION “G” Intranasal Suspension

One liter of a sterile aqueous suspension for intranasal instillation,containing 2 mg of an active ingredient in each milliliter, is preparedfrom the following types and amounts of ingredients: Active ingredient,micronized 1.5 g POLYSORBATE 80 5 g Methylparaben 2.5 g Propylparaben0.17 g Deionized water, q.s. 1000 ml.

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is aseptically filled intosterile containers.

The composition so prepared is useful for treating a neoplastic disease,by intranasal instillation of 0.2 to 0.5 ml given one to four times perday.

An active ingredient can also be present in the undiluted pure form foruse locally about the cutis, intranasally, pharyngolaryngeally,bronchially, or orally.

COMPOSITION “H” Powder

Five grams of an active ingredient in bulk form is finely divided bymeans of an air micronizer. The micronized powder is placed in ashaker-type container.

The foregoing composition is useful for treating a neoplastic disease,at localized sites by applying a powder one to four times per day.

COMPOSITION “I” Oral Powder

One hundred grams of an active ingredient in bulk form is finely dividedby means of an air micronizer. The micronized powder is divided intoindividual doses of 20 mg and packaged.

The foregoing powders are useful for treating a neoplastic disease, bythe oral administration of one or two powders suspended in a glass ofwater, one to four times per day.

COMPOSITION “J” Insufflation

One hundred grams of an active ingredient in bulk form is finely dividedby means of an air micronizer. The foregoing composition is useful fortreating a neoplastic disease, by the inhalation of 30 mg one to fourtimes a day. It is of course understood that such modifications,alterations and adaptations as will readily occur to the artisanconfronted with this disclosure are intended within the spirit of thepresent invention. TABLE I High Field (500 MHz) NMR Assignments forCribrostatin 6 (3),(3,8-dimethyl-9-ethoxy-imidazo[5,1-a]isoquinoline-7,10-dione) in CDCl₃¹H HMBC GOESY δ(mult, ¹³C j1xh = 140, mix = Position J, #H) and ¹⁵N jnxh= 8 COSY .3  1 8.29s, 1H 125.68 C-3  2  3 137.64  4  5 7.90d, 124.73C-3, C-6, H-6 H-6, 7.5Hz, 1H C-6a H-11  6 7.26d, 107.73 C-6a, C-7, H-57.5Hz, 1H C-10a  6a 125.00  7 184.86  8 130.06  9 156.16 10 180.58 10a123.49 10b 123.87 11 2.75s, 3H 12.57 C-3 H-5 12 2.06s, 3H 9.15 C-7, C-8,C-9 13 4.40q, 69.64 C-9, C-14 H-14 6.9Hz, 2H 14 1.41t, 15.97 C-13 H-136.8Hz, 3H N-4 189.5 N-2 273.9

TABLE II Cribrostatin 6 (3) Inhibitory Activity (GI₅₀, μg/ml) Against aPanel of Human Cancer Cell Lines and Mouse Leukemia Cell Type Cell LineCribrostatin-6 Pancreas-adenocarcinoma BXPC-3 >1 Breast-adenocarcinomaMCF-7 0.21 CNS Glioblastoma SF-268 0.24 Lung-NSC NCI-H460 >1Colon-adenocarcinoma KM20L2 >1 Prostate DU-145 0.38 Mouse Leukemia P3880.29

TABLE III Antimicrobial Activities of Cribrostatin 6. Minimum InhibitoryConcentration Microorganism (μg/ml) Candida albicans (ATCC 90028) 64Cryptococcus neoformans (ATCC 90112) 2 Micrococcus luteus (Presque Isle456) 16 Staphylococcus aureus (ATCC 29213) 16 Methicillin-resistant S.aureus 16 (clinical isolate) Enterococcus faecalis (ATCC 29212) 32Vancomycin-resistant E. faecalis 32 (clinical isolate) Bacillus subtilis(clinical isolate) 2 Streptococcus pneumoniae (ATCC 6303) 0.5Penicillin-resistant S. pneumoniae 2 (clinical isolate) Invasive S.pneumoniae (clinical isolate) 1 Group A Streptococcus (clinical isolate)16 Stenotrophomonas maltophilia (ATCC 13637) >64 Escherichia coli (ATCC25922) >64 Enterobacter cloacae (ATCC 13047) >64 Neisseria gonorrhoeae(ATCC 49226) 0.0625

1. A compound of the formula9-ethoxy-3,8-dimethyl-imidazo[5,1-a]isoquinoline-7,10-dione.
 2. Acompound having the following structure:


3. A compound having the following solid-state structure:


4. A compound characterized by having the following high field (500 MHz)NMR assignments in CDCl₃ ¹H HMBC GOESY δ(mult, ¹³C j1xh = 140, mix =Position J, #H) and ¹⁵N jnxh = 8 COSY .3  1 8.29s, 1H 125.68 C-3  2  3137.64  4  5 7.90d, 124.73 C-3, C-6, H-6 H-6, 7.5Hz, 1H C-6a H-11  67.26d, 107.73 C-6a, C-7, H-5 7.5Hz, 1H C-10a  6a 125.00  7 184.86  8130.06  9 156.16 10 180.58 10a 123.49 10b 123.87 11 2.75s, 3H 12.57 C-3H-5 12 2.06s, 3H 9.15 C-7, C-8, C-9 13 4.40q, 69.64 C-9, C-14 H-146.9Hz, 2H 14 1.41t, 15.97 C-13 H-13 6.8Hz, 3H N-4 189.5 N-2 273.9


5. A composition comprising the compound of claim 1 and apharmaceutically acceptable carrier therefor.
 6. A method for treatingneoplastic disease comprising administering a pharmaceutically effectiveamount of the compound of claim 1 to a human or animal subject.
 7. Amethod for treating bacterial infections comprising administering apharmaceutically effective amount of the compound of claim 1 to a humanor animal subject.
 8. A method for treating flngal infections comprisingadministering a pharmaceutically effective amount of the compound ofclaim 1 to a human or animal subject.
 9. A method for purifyingcribrostatin 6 by: (a) fractionating extract of blue marine spongeCribochalina sp. via successive elutions with CH₃OH, CH₂CL₂-CH₃OH (3:2),hexane-toluene-CH₃OH (3:1:1) and hexane/i-PrOH-CH₃OH (8:1:1), (b)triturating the blue fraction resulting from step (a) with lesspolarphase of the system hexane-EtOAc-CH₃OH-water (700:300:150:60) (c)filtering the solution and (d) obtaining a solid.